Polyketide synthases (PKSs) are a family of multi-enzyme assemblies that catalyze the synthesis of numerous structurally complex and biologically important natural products. Modular PKSs, such as the 6-deoxyerythronolide B synthase (DEBS), are a particularly interesting sub-class of PKSs that synthesize complex polyketides such as macrolides. Over the past decade, there has been considerable interest in studying these megasynthases, and in exploiting their modularity and broad substrate specificity for the engineered biosynthesis of "unnatural" natural products. Most products of modular PKSs are produced by relatively uncharacterized bacteria. As a result, every time a new natural product with promising biological properties is discovered, a considerable amount of time and expense must be incurred to obtain reliable quantities of the compound from natural sources, and an even greater investment is demanded before the biosynthetic pathway becomes amenable to rational engineering. An alternative is to develop robust and generally applicable technologies for the heterologous expression of polyketides in well-characterized microbial hosts. During the past proposal period, the metabolism of the model bacterium Escherichia coli was engineered to produce 6-deoxyerythronolide B (6dEB), the macrocyclic core of the antibiotic erythromycin. This engineered strain of E. coli harbors modifications in five endogenous genes; it also contains seven new genes from three different heterologous sources. The resulting cellular catalyst converts exogenous propionate into 6dEB in quantities approaching 200 mg/L over a 5-day process. During the next 3-year proposal period, we will focus on improving and extending the properties of E. coli as a host of choice for the biosynthesis of natural and unnatural polyketides. This will be accomplished through a combination of molecular biological tools, metabolic engineering strategies and fermentation technology development. The Specific Aims are: I] Engineering new pathways for precursor and product biosynthesis in E. coli; II] Improved fermentation protocols for enhancing polyketide productivity in E. coli; III] Further improvements in polyketide productivity of E. coli using functional genornic and metabolic engineering approaches; & IV] Heterologous production of two new complex natural products in E. coli. The implications of this research are 3-fold. First, given the availability of scalable protocols for fermenting E. coli to overproduce bioproducts, the ability to synthesize complex polyketides in this heterologous host will bode well for the practical production of these expensive bioactive natural products as well as their engineered derivatives. Second, the use of E. coli as a host for polyketide production opens the door for harnessing E. coli to engineer modular PKSs using directed and random approaches. Finally, the project is a good opportunity to train students at the interface of metabolic engineering & natural product biosynthesis.